Project Summary: Sleep is a widely conserved behavior and it is known to be regulated by changes in gene expression. However, the molecular basis of the regulation of sleep remains poorly understood. Research from our lab, and elsewhere, supports the idea that microRNAs (miRs) are involved. miRs are short non-coding RNA transcripts (20-24 bp in length) that target specific mRNAs, downregulating their expression. Results from a genetic screen in which miRs were downregulated by expression of transgenes which specifically bind particular miRs (miR-SPs), demonstrated that miR-190 is involved in Drosophila sleep regulation. Pan- neuronal expression of miR-190-SP or mutation of the miR-190 gene both elicited dramatic changes in Drosophila sleep behavior, including decreased and fragmented total sleep, as well as deficient sleep homeostasis. Expression of miR-190-SP in limited numbers of cells in different brain regions using the Gal4/UAS system showed that disruption of miR-190 function must occur in a large number of neurons to affect Drosophila sleep regulation. At the molecular level, our preliminary data from RNA seq of adult heads showed that pan-neuronal expression of miR-190-SP induces an up or downregulation of multiple genes, including 9 genes which are intimately involved in dopamine (DA) signaling, the major pro-arousal system of the fly. Temporally-controlled expression of miR-190-SP demonstrated that the full miR-190 knockdown phenotype requires expression during the middle of pupation; reduction of miR-190 only in earlier developmental stages produced no phenotype. Adult-specific expression minimally affected sleep time, but disrupted homeostasis. Taken together, our data suggest that miR-190 functions during development to specify the activity of the adult arousal system and has an on-going role in adult sleep homeostasis. This proposal aims to unravel the developmental role of miR-190 in the establishment of adult sleep behaviors, dissect the involvement of dopaminergic signaling in this regulation of sleep, and discover the cellular locus of miR-190's role in homeostasis. First, I will identify the molecular targets of miR-190 by performing of Ago-IP RNAseq and validate those by means of behavioral experiments. Additionally, I will pursue the bases of miR-190 adult phenotype and its regulation by dopaminergic signaling. I will study the morphology of DA cells in these miR-190-deficient genotypes to determine if their sleep phenotype is caused by changes in cell number or connectivity. By using genetically encoded functional CaLexA imaging sensor I will look at the activity of DA cells in miR-190 hypomorph flies and determine if they are hyperactive, compared to control flies. Lastly, I will characterize miR-190's spatiotemporal pattern of expression for sleep homeostasis regulation by expressing a miR-190 sponge in brain areas known to affect sleep homeostasis and designing a miR-190 sensor capable of reading out miR- 190 levels. These experiments will shine light on the role of miRs in modulation of sleep and will be critical for the understanding of normal sleep, as well as pathological sleep conditions.